The present application claims priority to German Patent Application No. 198 13 317.0, filed Mar. 26, 1998.
1. Field of the Invention
The invention provides an improved method of xe2x80x9cwhole genome amplificationxe2x80x9d (WGA) that is suitable for performing DNA analysis starting with just one or a few cells. The improvement of the method basically lies in the fact that, to amplify the DNA, a mixture of two DNA polymerases is used, at least one of which possesses 3xe2x80x2-5xe2x80x2 exonuclease activity.
2. Description of Related Art
WGA methods are especially important in the field of differential tumor diagnostics. The goal of differential tumor diagnostics at the molecular level is to analyze nucleic acid samples from single cells or cell populations that contain no non-malignant cells (Emmert-Buck et al., 1996; Bxc3x6hm and Wielang, 1997). To obtain the appropriate samples, therefore, cell sorting methods (Abeln et al., 1994, Barret et al., 1996), microdissection methods (Shibata et al., 1992, Shibata et al., 1993, Emmert-Buck et al., 1994, Noguchi et al., 1994, Zhuang et al., 1995, Bxc3x6hm et al., 1997), and laser microdissection (Schxc3xctze and Clement-Sengewald, 1994) are used with increasing frequency.
Especially sensitive methods of nucleic acid amplification are required before a molecular analysis of single cells or populations of a few cells can be performed, however. According to the prior art, methods of xe2x80x9cwhole genome amplificationxe2x80x9d (WGA) are especially well-suited for this application. These are methods that comprise two consecutive amplification reactions. The first amplification reaction is carried out using randomized primers, and the second amplification is carried out using specific primers. WGA can be used, for instance, to diagnose hereditary diseases as part of pre-implantation diagnostic testing of biopsied blastomere cells (Kristianson et al., 1994, Snabes et al., 1994, van der Veyer et al., 1995), or as part of prenatal diagnostic testing of nucleated erythrocytes in maternal blood (Sekizawa et al., 1996).
According to the prior art, WGA is usually used to analyze microsatellites in tumor biopsies to detect microsatellite instability or the loss of heterozygosity. According to the prior art, the analyzed sample must contain so many cells, however, that a quantitatively disproportionate amplification of individual alleles caused by accidental preparation artifacts is ruled out (Zhang et al., 1992, Barret et al., 1995, Cheung and Nelson, 1996, Faulkner and Leigh, 1998). For instance, a batch of about 1,000 cells was investigated in a microsatellite analysis of FACS-sorted aneuploidal esophageal tumor cells (Barret et al., 1995).
In contrast to conventional in situ hybridization (Van Ommen et al., 1995) or conventional, specific PCR (Becker et al., 1996), multiple analyses of the same sample can be carried out using WGA methods. Two different methods are used. In xe2x80x9cdegenerate oligonucleotide primer PCRxe2x80x9d (DOP-PCR), amplification primers with defined sequences on the 5xe2x80x2 and 3xe2x80x2 ends and a randomized hexamer region in the middle of the primer are used (Telenius et al., 1992). Starting with only slightly stringent conditions in the first 5 thermal cycles, the next 35 thermal cycles are carried out under more stringent conditions at a higher annealing temperature so that, during these cycles, only completely complementary primers can bind to the DNA to be amplified. These methods are used, for instance, as the first step before performing an in situ hybridization with flow-sorted chromosomes (Blennow et al., 1992; Telenius et al., 1992; Kallionemie et al., 1994), or to perform comparative genomic hybridization (CGH) (Du-Manoir et al., 1993; Schlegel et al., 1995).
An alternative principle of WGA is xe2x80x9cprimer-extension preamplificationxe2x80x9d (PEP-PCR, Zhang et al., 1992). In contrast to the DOP-PCR, this method uses completely randomized 15-mer amplification primers. During 50 consecutive thermal cycles, denaturing is first carried out at 92xc2x0 C., followed by hybridization under only slightly stringent temperature conditions at 37xc2x0 C. This temperature is increased successively to 55xc2x0 C. at a rate of about 0.1xc2x0 C./second. At this temperature, the polymerase extension reaction takes place for another 4 minutes.
All methods known in the prior art (von Eggeling and Spielvogel, 1995) have the disadvantage of insufficient sensitivity, however, because a relatively large number of cells must be used to increase the possibility of obtaining an amplification product. In addition, the sensitivity of the assay is reduced even more as the length of the fragment to be amplified increases. For this reason, the methods known in the prior art had only been used in the amplification of relatively small fragments with a length of up to 580 base pairs (Snabes et al., 1994).
Another main disadvantage of the PEP-PCR known in the prior art lies in the fact that a convincing DNA mutation analysis has never been reliably carried out due to the inherent error rate of the Taq polymerase used. The error rate is due to the fact that using Taq polymerase during amplification leads to AT/GC transitions in the amplification product (Keohvong and Thily, 1989). In addition, deletion mutations may arise when Taq polymerase is used if the DNA to be amplified is capable of forming secondary structures (Carriello et al., 1991). The risk of obtaining amplification artifacts is especially high with WGA, however, because more than 80 amplification cycles are usually carried out during the 2 to 3 amplification reactions.
To avoid sequence artifacts during nucleic acid amplification, the use of DNA polymerases with 3xe2x80x2-5xe2x80x2 exonuclease activity was also known in the prior art (Flaman et al., 1994; Casas and Kirkpontrick, 1996). The use of polymerases without 3xe2x80x2-5xe2x80x2 exonuclease activity for WGA, however, leads to a further reduction in the sensitivity of the method, because such polymerases possess much less processivity than Taq DNA polymerases, for instance. As a result, the products created during preamplification when randomized primers are used are not long enough to serve as matrices for the subsequent specific PCR reaction if the fragment to be amplified exceeds a certain size.
The technical object to be solved with this invention was therefore to develop a method with which, starting with the smallest possible number of cells, specific nucleic acid fragments of high quality, i.e., containing no sequence artifacts, could be amplified and then analyzed. The quality of the amplification products should make it possible to carry out reliable mutation analyses, sequence analyses, and unequivocally interpretable microsatellite analyses. This objective is solved by an improved method of primer-extension preamplification (PEP-PCR, Zhang et al., 1992).
Object of the invention is therefore a method for the amplification of nucleic acid fragments from a sample comprising two or three thermocyclic amplification reactions in which completely randomized primers are used in the first amplification reaction and specific primers are used in the second amplification reaction, characterized in that, to amplify the DNA, a mixture of at least two DNA polymerases is used, at least one of which possesses 3xe2x80x2-5xe2x80x2 exonuclease activity. This characteristic is also called proofreading activity in the context of polymerases (Flaman et al., 1994).
An amplification reaction comprises about 20 to 60 thermal cycles. The first amplification reaction preferably comprises at least 40 thermal cycles and, most preferably, at least 50 thermal cycles. The second amplification reaction preferably comprises at least 30 thermal cycles, and most preferably, at least 40 thermal cycles.
Each thermal cycle comprises a denaturing phase, an annealing phase, and at least one elongation phase. Denaturation into single strands preferably takes place at temperatures of between 90xc2x0 C. and 96xc2x0 C. The annealing phase to hybridize the primers with the target nucleic acid preferably takes place at temperatures of between 30xc2x0 C. and 50xc2x0 C. Most preferably, the annealing phase takes place at temperatures of between 35xc2x0 C. and 45xc2x0 C. During the first amplification reaction, the annealing phase most preferably takes place at about 37xc2x0 C. The elongation phase is carried out at temperatures of between 50xc2x0 C. and 75xc2x0 C. In a preferred embodiment, the elongation phase of the first amplification reaction takes place at temperatures of between 50xc2x0 C. and 60xc2x0 C. A temperature of about 55xc2x0 C. is especially preferred.
An advantage of the embodiment of the invention claimed is a slow transition between the annealing phase and the elongation phase. This transition is carried out at a speed of less than 0.5xc2x0 C. per second. It is most advantageous for the temperature transition to be carried out at a speed of 0.1 xc2x0 C./second.
To perform the method provided by this invention, it is advantageous for the elongation to be carried out during the first amplification reaction in the majority of cycles using two or more elongation steps, with one elongation carried out at a lower temperature and then continuing the elongation at a higher temperature. Using this approach, populations of especially long amplicons are created during the first amplification reaction. In this embodiment, the first amplification reaction preferably takes place at about 55xc2x0 C., and the second amplification reaction takes place at about 65xc2x0 C. to 72xc2x0 C. A temperature of about 68xc2x0 C. is optimal.
The primers used in the first amplification reaction are completely randomized, i.e., a population of single-stranded oligonucleotides is used in which every single nucleotide on every single position can comprise one of four nucleotide components A, T, G, or C. These primers are preferably 10-20 nucleotides long. Most preferably, the primers are about 15 nucleotides long. The specific primers used in the second amplification reaction are characterized in that they have a sequence that is identical to a sequence of the target nucleic acid or its complementary sequence over a range of at least 10 nucleotides. The specific primers used to carry out a xe2x80x9cnested PCRxe2x80x9d in a potential third amplification reaction are selected according to the same criteria as the primers used in the second amplification reaction. The sequences of the primers used that are identical to the target nucleic acid or its complement must be a component of the sequence amplified in the second amplification reaction.
The mixture of DNA polymerases provided by the invention preferably contains a thermostable DNA polymerase without 3xe2x80x2-5xe2x80x2 exonuclease activity such as Taq DNA polymerase, for instance, and another thermostable DNA polymerase with 3xe2x80x2-5xe2x80x2 exonuclease activity, such as Pwo DNA polymerase obtained from Pyrokokkus woesii (Boehringer Mannheim order no. 1644947). Other DNA polymerases without 3xe2x80x2-5xe2x80x2 exonuclease activity can also be used as a component of the polymerase mixture.
One of the embodiments of the method claimed is a method for DNA amplification. To ensure the sensitivity of detecting certain sequences, it is advantageous to carry out the cell analysis of the material to be analyzed using enzymatic protease digestion to obtain the sample DNA. Proteinase K can be used, for instance.
In another embodiment of the method claimed, RNA is first isolated from the physical material to be analyzed. The sample of physical material can comprise one cell, fewer than 10 cells, or fewer than 100 cells. To obtain RNA, it is preferable to use chemical lysis using buffers that contain guanidinum isothiocynate. A corresponding cDNA is then created using a reverse transcriptase reaction. This cDNA is then used as the starting material for the primer-extension preamplification provided by this invention. The cDNA is preferably obtained via reverse transcription of poly-A RNA.
The methods claimed are suitable for use in analyzing samples from physical material that comprises just one or a few cells. The method claimed is therefore especially well-suited for use in analyzing nucleic acids from tissue slices. Such tissue slices can be obtained from frozen, formalin-fixed, or paraffin-embedded material. An appropriate protease digestion step is especially advantageous for these embodiments.
The use of polymerase mixtures in the primer-extension preamplification PCR provided by this invention leads to a surprisingly high sensitivity of DNA detection that cannot be achieved using the methods known from the prior art. Object of the invention is therefore a method for the amplification of nucleic acid fragments comprising two or three thermocyclic amplification reactions. Completely randomized primers are used in the first amplification reaction and specific primers are used in the second amplification reaction. In addition, the sample contains a quantity of nucleic acid corresponding to an equivalent of no more than 100 cells. This invention is characterized in that the likelihood of the amplificates forming is greater than 90%. Object of the invention are especially methods with which the likelihood is greater than 90% that amplificates will form from an equivalent of no more than 5-10 cells. In a special embodiment, the likelihood of amplificates forming from the equivalent of one cell is greater than 50%.
The method provided by the invention is suitable for use in the amplification of nucleic acid fragments having a length between 100 and 1000 base pairs. The method is especially suited for use in the amplification of nucleic acid fragments having a length between 150 and 550 base pairs.
In summary, the method provided by the invention makes it possible to amplify specific DNA fragments from nucleic acid samples obtained from just one or a few cells while ruling out or at least minimizing the creation of amplification artifacts. Object of the invention therefore also includes the use of DNA amplified according to the invention in mutation analysis. The mutation analysis can be carried out in a special embodiment in that the nucleic acid fragment amplified according to the invention is analyzed using a sequencing reaction.
Object of the invention claimed is also the use of DNA amplified according to the invention to analyze microsatellites, and especially to analyze nucleic acid samples obtained from frozen, formalin-fixed, or paraffin-embedded tissue slices. Cell equivalents of 5-20 cells are preferably used in this embodiment, because an even amplification of alleles from the same gene locus is not ensured if smaller quantities of cells are used. The analysis of microsatellites according to the invention can be used to diagnose microsatellite instability and the loss of heterozygosity (Boehm and Wieland, 1997).
In addition, when the equivalent of at least 10 cells is used, the likelihood of obtaining amplification products of both alleles is greater than 90%.